Mammalian tooth development is regulated by dynamic interactions among many molecules encoded either by globally expressed genes or tooth-specific genes regulated in a spatiotemporal manner. The tooth is a unique organ that develops through a number of morphological and cytological changes leading to four structurally and functionally distinct components: enamel, dentin, cementum, and dental pulp. Dentin, enamel, and cementum are highly mineralized and provide strength to fully formed teeth, whereas dental pulp provides constant metabolic support. Ameloblasts secrete several proteins to form the enamel extracellular matrix (ECM). Amelogenins constitute more than 90% of secreted enamel ECM proteins and are believed to play an important role in the biomineralization of enamel. Odontoblasts secrete dentin ECM, which contains several collagenous and noncollagenous proteins and forms predentin. The predentin mineralizes to form mature dentin. Dentin sialophosphoprotein (DSPP), one of the major noncollagenous proteins, has been considered to be a key regulator in dentin formation. Genetic mutations in amelogenin and DSPP genes are implicated in the most common genetic disorders of enamel and dentin. TGF-beta1 is expressed throughout tooth development and regulates the synthesis of ECM proteins and adhesion molecules. TGF-beta also modulates immune responses and wound healing. We have used the powerful research tool of mouse molecular genetics to study the developmental roles of the amelogenin, ameloblastin, DSPP, and TGF-beta1 genes in the tooth. Our broad hypothesis is that each of these genes plays a unique role in tooth development, and their crosstalk orchestrates the tooth mineralization process. We have used a variety of molecular approaches, such as conventional and conditional gene targeting, genomic and proteomic analysis, and microarray screening, to investigate the crosstalk amongst these genes. With the help of expert collaborators, we have also utilized special techniques such as scanning and transmission electron microscopy to analyze the composition and nanoindentation of teeth to determine bonding and tensile strength. Our present studies will not only contribute to a greater understanding of the molecular roles of these candidate genes in tooth development, but should also aid future efforts toward the development of more effective treatments for tooth disorders.[unreadable] [unreadable] Enamel matrix is secreted by ameloblasts and mineralizes to form enamel. Amelogenins are major constituents of the enamel matrix and are believed to play an important role in enamel mineralization. Mutations in the human amelogenin gene have been reported in amelogenesis imperfecta patients. We generated amelogenin-null mice, which displayed a typical X-linked amelogenesis imperfecta phenotype characterized by chalky white teeth, enamel hypoplasia, a lack of prismatic crystals, and cuspal attrition. Elemental analysis indicated that the enamel contained normal hydroxyapatite crystals, confirming the continuation of mineralization in the absence of the amelogenins. These results establish that amelogenins are essential for the organization of the crystal pattern and enamel development but are not required for initiation of mineral crystal formation. Amelogenin proteins are products of RNA splicings and we evaluated some of these products for their precise function by breeding the null mice with transgenic mice overexpressing the shortest amelogenin peptide, LRAP, to assess its effects on the amelogenin-null phenotype. These double-transgenic mice failed to rescue the tooth defects seen in the amelogenin-null mice, indicating the importance of functional differences in amelogenin splice variants. In addition to their enamel-specific roles, amelogenins are also implicated in the formation of root cementum. During cementogenesis, Hertwig?s epithelial root sheath dissociiates to form cell aggregates (epithelial rests of Malassez) that are located between the alveolar bone and the root sheath. The mesenchyme-derived cementoblasts secrete cementum matrix onto the root surface to form cementum. The presence of amelogenins was reported earlier on the root surface close to the site of extracellular cementum and in the epithelial remnants of the root sheath. Interestingly, our recent studies discovered that two amelogenin splice variants, M180 and LRAP, are predominantly expressed in mouse tooth roots. Additionally also, we have identified LRAP expression by RT PCR from brain, eye, and calvaria tissue. Thus, our studies clearly demonstrate that the amelogenin splice variants are expressed in a nonenamel component of the tooth, namely tooth roots, thereby implying additional roles. [unreadable] [unreadable] In order to determine the precise role of amelogenins in tooth roots, we carefully analyzed tooth roots of aging amelogenin-null mice. This analysis unexpectedly revealed progressive cementum defects in the null mice. The cementum of the null mice displayed resorptive lacunae at sites where periodontal ligaments attach to the cementum surface. Multiple intrusive attachments of periodontal ligament cells extended through the cementum into the root dentin of the null mice. The aim of our ongoing study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament (CM/PDL) cells. Cocultures of wild-type (WT) osteoclast progenitor (OP) and amelogenin-null (KO) CM/PDL cells displayed an increased number of tartrate-resistant acid phosphatase (TRAP)-positive cells as compared to the cocultures of WT-OP + WT-CM/PDL cells. The addition of LRAP to both the cocultures significantly reduced the number of TRAP-positive cells. RANKL expression in the CM/PDL cell cultures was decreased by the addition of LRAP but not P172, a porcine homolog of mouse M180. Proliferation and migration rates of the KO-CM/PDL cells were lower as compared to WT cells and increased with the addition of either LRAP or P172. Our data suggest LRAP inhibits osteoclastogenesis and that both P172 and LRAP promote periodontal cell proliferation and migration of CM/PDL cells.[unreadable] [unreadable] The structural tooth defects observed in the DSPP-/- mice were enlarged pulp chambers, increased width of predentin zone, hypomineralization, pulp exposure, an irregular mineralization front, and a lack of uniform coalescence of calcospherites in the dentin. The levels of the proteoglycans biglycan and decorin were increased in the widened predentin zone and in the void spaces among the calcospherites in the null dentin. These enhanced levels correlated well with the regions defective in mineralization. Their precise role is currently being analyzed using double knockout mice for DSPP/decorin, and DSPP/biglycan. We have begun to analyze specific roles of DSPP cleavage products, DSP and DPP. [unreadable] [unreadable] TGF-beta1 is a key regulator of many cellular processes, including cell adhesion, immune response and synthesis of extracellular matrix proteins. We characterized the enamel defects in a transgenic mouse model overexpressing TGF-beta1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice developed distinct dentin defects similar to those seen in human tooth disorders. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. Our ongoing studies are focused on evaluating precise roles of TGF-beta isoforms and their signaling pathways in development and disease.[unreadable] [unreadable] We are continuing our efforts to characterize craniofacial and skeletal structures in mouse models for Fabry and Mucolipidosis-IV diseases.